Systematic Identification of Cellular Regulators of Gammaherpesvirus Lytic Replication

Gammaherpesviruses are clinically important human pathogens associated with multiple types of tumors. The life cycle of gammaherpesviruses consists of two distinct phases, latency and lytic replication, both of which are important for the viral-associated pathogenesis. Murine gammaherpesvirus 68 (MHV-68) has been used as a model to study virus-host interactions of human gammaherpesviruses because of its genomic and biological similarities. During the course of MHV-68 lytic infection, the virus is highly dependent on cellular machinery to facilitate its replication. Host cells, on the other hand, develop mechanisms to sense and restrict virus replication. Here, we present an integrative systems approach to elucidate the interactions between MHV-68 and its host. Our previous yeast-two-hybrid analysis and genome-wide expression profiling implicated hundreds of cellular factors mediating virus-host interactions. Among the cellular factors identified, those involved in kinase- and transcription factor- regulated signaling are the most highly enriched. Therefore, high-throughput screens of cellular kinome and transcription factors cDNA and siRNA libraries were carried out, which uncovered 125 MHV-68 dependency factors and 213 restriction factors. We found that many cellular factors affect MHV-68 replication by regulating RTA transcription. Characterization of this subset of genes showed they also play roles in KSHV latency maintenance and reactivation. Furthermore, cellular factors regulating later steps of viral replication, such as viral late gene expression and egress, were also identified and validated. Finally we compared the viral restriction factors to the results of a kinome siRNA screen designed to identify cellular regulators of innate immune response to cytosolic DNA. Remarkably, 23 genes were found among the top 10% of the hits of both screens. The MHV-68 restriction factor RIOK3 was confirmed to play a role in IRF3 activation and type I interferon induction mediated by both cytosolic DNA and RNA. RIOK3 is required for cellular antiviral response to both DNA virus (MHV-68) and RNA virus (Influenza A), suggesting a central role in innate immunity. Further analysis revealed that RIOK3 complexes with TBK1 and IRF3, bridging the activity between the two. Collectively, this multilayered approach unfolded a comprehensive map of gammaherpesvirus-host interactions which will provide basis for developing new anti-viral therapies